Limits...
The AEROPATH project targeting Pseudomonas aeruginosa: crystallographic studies for assessment of potential targets in early-stage drug discovery.

Moynie L, Schnell R, McMahon SA, Sandalova T, Boulkerou WA, Schmidberger JW, Alphey M, Cukier C, Duthie F, Kopec J, Liu H, Jacewicz A, Hunter WN, Naismith JH, Schneider G - Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. (2012)

Bottom Line: A major concern is Gram-negative bacteria, for which the discovery of new antimicrobial drugs has been particularly scarce.The crystal structures of eight of these targets, ranging from hypothetical unknown proteins to metabolic enzymes from different functional classes (PA1645, PA1648, PA2169, PA3770, PA4098, PA4485, PA4992 and PA5259), are reported here.The structural information is expected to provide a firm basis for the improvement of hit compounds identified from fragment-based and high-throughput screening campaigns.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biomedical Sciences Research Complex, University of St Andrews, St Andrews KY16 9ST, Scotland.

ABSTRACT
Bacterial infections are increasingly difficult to treat owing to the spread of antibiotic resistance. A major concern is Gram-negative bacteria, for which the discovery of new antimicrobial drugs has been particularly scarce. In an effort to accelerate early steps in drug discovery, the EU-funded AEROPATH project aims to identify novel targets in the opportunistic pathogen Pseudomonas aeruginosa by applying a multidisciplinary approach encompassing target validation, structural characterization, assay development and hit identification from small-molecule libraries. Here, the strategies used for target selection are described and progress in protein production and structure analysis is reported. Of the 102 selected targets, 84 could be produced in soluble form and the de novo structures of 39 proteins have been determined. The crystal structures of eight of these targets, ranging from hypothetical unknown proteins to metabolic enzymes from different functional classes (PA1645, PA1648, PA2169, PA3770, PA4098, PA4485, PA4992 and PA5259), are reported here. The structural information is expected to provide a firm basis for the improvement of hit compounds identified from fragment-based and high-throughput screening campaigns.

Show MeSH

Related in: MedlinePlus

(a) Schematic view of the structure of PA2169, a protein of unknown function. The dotted line indicates the flexible loop that is not well defined in electron density. (b) Surface representation of PA2169 with residues colour-coded according to sequence conservation from white (not conserved) to cyan (invariant). (c) View of the potential metal-binding site in PA2169 comprising residues Glu102, Asp106 and His139.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3539698&req=5

fig2: (a) Schematic view of the structure of PA2169, a protein of unknown function. The dotted line indicates the flexible loop that is not well defined in electron density. (b) Surface representation of PA2169 with residues colour-coded according to sequence conservation from white (not conserved) to cyan (invariant). (c) View of the potential metal-binding site in PA2169 comprising residues Glu102, Asp106 and His139.

Mentions: The structure of PA2169 revealed a four-helix-bundle fold (Fig. 2 ▶a and Supplementary Fig. S1b) with the same topology as observed in the ferritin-like module of the redox-defence protein from Mycobacterium smegmatis (Roy et al., 2007 ▶). The crystal structure is thus consistent with circular-dichroism spectroscopy using purified protein samples, which suggested an all-α fold for PA2169. A disulfide bond is formed between Cys30 and Cys101. The protein lacks the metal-ion-binding site that is typical of the ferritin family (Theil, 2011 ▶) and the iron-binding residues are not conserved. In the structure of PA2169 a triad composed of Glu102, Asp106 and His139 is found in a different location to the iron-binding site in the ferritin family and resembles a potential metal-binding site. These residues are conserved in proteins belonging to the DUF2383 domain sequence family (Figs. 2 ▶b and 2 ▶c). However, attempts to provide experimental evidence for metal binding using differential scanning fluorimetry and cocrystallization were unsuccessful.


The AEROPATH project targeting Pseudomonas aeruginosa: crystallographic studies for assessment of potential targets in early-stage drug discovery.

Moynie L, Schnell R, McMahon SA, Sandalova T, Boulkerou WA, Schmidberger JW, Alphey M, Cukier C, Duthie F, Kopec J, Liu H, Jacewicz A, Hunter WN, Naismith JH, Schneider G - Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. (2012)

(a) Schematic view of the structure of PA2169, a protein of unknown function. The dotted line indicates the flexible loop that is not well defined in electron density. (b) Surface representation of PA2169 with residues colour-coded according to sequence conservation from white (not conserved) to cyan (invariant). (c) View of the potential metal-binding site in PA2169 comprising residues Glu102, Asp106 and His139.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539698&req=5

fig2: (a) Schematic view of the structure of PA2169, a protein of unknown function. The dotted line indicates the flexible loop that is not well defined in electron density. (b) Surface representation of PA2169 with residues colour-coded according to sequence conservation from white (not conserved) to cyan (invariant). (c) View of the potential metal-binding site in PA2169 comprising residues Glu102, Asp106 and His139.
Mentions: The structure of PA2169 revealed a four-helix-bundle fold (Fig. 2 ▶a and Supplementary Fig. S1b) with the same topology as observed in the ferritin-like module of the redox-defence protein from Mycobacterium smegmatis (Roy et al., 2007 ▶). The crystal structure is thus consistent with circular-dichroism spectroscopy using purified protein samples, which suggested an all-α fold for PA2169. A disulfide bond is formed between Cys30 and Cys101. The protein lacks the metal-ion-binding site that is typical of the ferritin family (Theil, 2011 ▶) and the iron-binding residues are not conserved. In the structure of PA2169 a triad composed of Glu102, Asp106 and His139 is found in a different location to the iron-binding site in the ferritin family and resembles a potential metal-binding site. These residues are conserved in proteins belonging to the DUF2383 domain sequence family (Figs. 2 ▶b and 2 ▶c). However, attempts to provide experimental evidence for metal binding using differential scanning fluorimetry and cocrystallization were unsuccessful.

Bottom Line: A major concern is Gram-negative bacteria, for which the discovery of new antimicrobial drugs has been particularly scarce.The crystal structures of eight of these targets, ranging from hypothetical unknown proteins to metabolic enzymes from different functional classes (PA1645, PA1648, PA2169, PA3770, PA4098, PA4485, PA4992 and PA5259), are reported here.The structural information is expected to provide a firm basis for the improvement of hit compounds identified from fragment-based and high-throughput screening campaigns.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biomedical Sciences Research Complex, University of St Andrews, St Andrews KY16 9ST, Scotland.

ABSTRACT
Bacterial infections are increasingly difficult to treat owing to the spread of antibiotic resistance. A major concern is Gram-negative bacteria, for which the discovery of new antimicrobial drugs has been particularly scarce. In an effort to accelerate early steps in drug discovery, the EU-funded AEROPATH project aims to identify novel targets in the opportunistic pathogen Pseudomonas aeruginosa by applying a multidisciplinary approach encompassing target validation, structural characterization, assay development and hit identification from small-molecule libraries. Here, the strategies used for target selection are described and progress in protein production and structure analysis is reported. Of the 102 selected targets, 84 could be produced in soluble form and the de novo structures of 39 proteins have been determined. The crystal structures of eight of these targets, ranging from hypothetical unknown proteins to metabolic enzymes from different functional classes (PA1645, PA1648, PA2169, PA3770, PA4098, PA4485, PA4992 and PA5259), are reported here. The structural information is expected to provide a firm basis for the improvement of hit compounds identified from fragment-based and high-throughput screening campaigns.

Show MeSH
Related in: MedlinePlus